THE PROTEOMIC PROFILE OF CELLS REPROGRAMMED USING HUMAN EMBRYONIC CARCINOMA CELL EXTRACTS

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PEWSEY E.; BRUCE C.; GEORGIOU A.S.; JONES M.; BAKER D.; OW S.Y.; WRIGHT P.C.; FREBERG C.; COLLAS P.; FAZELI A.

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Journal: International Journal of Reproductive BioMedicine Year:2009 | Volume:7 | Issue:SUPPL 2 Page(s): 0-0

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Title

THE PROTEOMIC PROFILE OF CELLS REPROGRAMMED USING HUMAN EMBRYONIC CARCINOMA CELL EXTRACTS

Author(s)

Keywords

NUCLEAR REPROGRAMMING

FLOW CYTOMETRIC ANALYSIS

EMBRYONIC CELL SURFACE MARKERS

2D-PAGE

DEDIFFERENTIATION

Abstract

Introduction: The reversal of the differentiated state of somatic nuclei is a phenomenon known as NUCLEAR REPROGRAMMING. The functional reprogramming of a differentiated cell to a pluripotent state presents beneficial applications in regenerative medicine. Our study aimed to characterise the proteomic profile of 293T cells (human embryonic kidney epithelial cell line) that were subjected to a reprogramming protocol using undifferentiated embryonic carcinoma (NCCIT) cellular extracts.Materials and Methods: 293T cells were reversibly permeabilised using the bacterial toxin Streptolysin O. Permeabilised cells were incubated in an extract of NCCIT cells (293T+Nex) or 293T cells (293T+293Tex) for 1 hr at 37oC. Cells were resealed with CaCl2 and further cultured.Results: Approximately 20- and 2-fold increases in Oct4 and Sox2 gene expression respectively were observed by real-time RT-PCR in 293T+Nex cells compared to 293T cells. However, there was no alteration in DNMT3B gene expression. Karyotyping confirmed that the 293T+Nex cells were of 293T origin, indicating that increased Oct4 and Sox2 expression was not the result of contaminating NCCIT cells. FLOW CYTOMETRIC ANALYSIS revealed that approximately 20% of the 293T+Nex cells were positive for SSEA-4 but expressed no other cell surface antigens associated with pluripotency. 2D PAGE revealed approximately 400 protein spots in the proteomic profile of each cell type studied, with the proteomic profiles of 293T+Nex and 293T+293Tex being compared to 293T and NCCIT. Protein spots displaying greater than a±1.5-fold alteration in expression were considered to be altered. At least 10 protein spots in the 293T+Nex proteome had a similar expression profile to NCCIT while remaining unaltered in 293T+293Tex (control). We suspect that these proteins are likely to be involved in conferring, or are the consequence of, NUCLEAR REPROGRAMMING of 293T+Nex cells from a differentiated state to a pluripotent state.Conclusion: Using mass spectrometry we identified these proteins which among others included 78 kDa glucose-regulated protein precursor and Tropomyosin alpha-3 chain. Our present investigation provides evidence that proteins are altered in a specific manner in 293T cells subjected to a reprogramming protocol using embryonic carcinoma cell extracts.

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APA: Copy

PEWSEY, E., BRUCE, C., GEORGIOU, A.S., JONES, M., BAKER, D., OW, S.Y., WRIGHT, P.C., FREBERG, C., COLLAS, P., & FAZELI, A.. (2009). THE PROTEOMIC PROFILE OF CELLS REPROGRAMMED USING HUMAN EMBRYONIC CARCINOMA CELL EXTRACTS. INTERNATIONAL JOURNAL OF REPRODUCTIVE BIOMEDICINE (IRANIAN JOURNAL OF REPRODUCTIVE MEDICINE), 7(SUPPL 2), 0-0. SID. https://sid.ir/paper/295437/en

Vancouver: Copy

PEWSEY E., BRUCE C., GEORGIOU A.S., JONES M., BAKER D., OW S.Y., WRIGHT P.C., FREBERG C., COLLAS P., FAZELI A.. THE PROTEOMIC PROFILE OF CELLS REPROGRAMMED USING HUMAN EMBRYONIC CARCINOMA CELL EXTRACTS. INTERNATIONAL JOURNAL OF REPRODUCTIVE BIOMEDICINE (IRANIAN JOURNAL OF REPRODUCTIVE MEDICINE)[Internet]. 2009;7(SUPPL 2):0-0. Available from: https://sid.ir/paper/295437/en

IEEE: Copy

E. PEWSEY, C. BRUCE, A.S. GEORGIOU, M. JONES, D. BAKER, S.Y. OW, P.C. WRIGHT, C. FREBERG, P. COLLAS, and A. FAZELI, “THE PROTEOMIC PROFILE OF CELLS REPROGRAMMED USING HUMAN EMBRYONIC CARCINOMA CELL EXTRACTS,” INTERNATIONAL JOURNAL OF REPRODUCTIVE BIOMEDICINE (IRANIAN JOURNAL OF REPRODUCTIVE MEDICINE), vol. 7, no. SUPPL 2, pp. 0–0, 2009, [Online]. Available: https://sid.ir/paper/295437/en

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